Factor XIII (FX III) is a zymogen in blood plasma that is transformed into its active form (FXIIIa) by thrombin and Ca.sup.2+. FXIIIa [also called activated fibrin-stabilizing factor, fibrinoligase, or plasma transglutaminase, EC 2.3.2.13(R-glutaminylpeptide:amine .gamma.-glutamyltransferase)] is the last enzyme generated in the blood coagulation cascade and catalyzes the formation of intermolecular .gamma.-glutamyl-.epsilon.-lysine crosslinks between fibrin molecules, thereby stabilizing the fibrin clot structure and conferring resistance to fibrinolysis. The crosslink formation by FXIIIa is important for normal blood coagulation and wound healing, and possibly in pathologic conditions such as thrombosis, atherosclerosis, and tumor growth and metastasis.
For clinical and basic studies on FXIII, a number of assays have been developed for measuring enzyme activity generated after treatment of the inactive zymogen with thrombin and Ca.sup.2+.
Lorand et al., "Diagnostic and Genetic Studies on Fibrin-Stabilizing Factor with a New Assay Based on Amine Incorporation," 48 J. Clinical Investigation 1054 (1969) have described an assay in which fibrinoligase catalyzes the incorporation of a fluorescent amine, monodansylcadaverine into casein. A fluorimetric method is then employed to analyze the reaction product. Curtis et al., "Fibrin-Stabilizing Factor (Factor XIII)" in 45 Methods in Enzymology 177-191 (1976), describe several methods for assaying Factor XIII including an assay utilizing the incorporation of dansylcadaverine into casein followed by measuring protein-bound fluorescence, a continuous rate assay for measuring enzymic incorporation of a fluorescent amine into a protein acceptor, a filter paper assay for measuring enzymic incorporation of radioactively labeled amines into proteins, a continuous rate assay for measuring the production of free thiol, and a continuous rate assay for measuring water-insoluble fluorescent amide. Folk et al., "Transglutaminases" in 113 Methods in Enzymology 358, 364 (1982), describe an assay which measures incorporation of radiolabeled putrescine into a casein mixture. Miraglia et al., 144 Analytical Biochemistry 165-171 (1985) describe Factor XIIIa measurement by incorporation of [.sup.3 H] putrescine into casein. Lorand et al., 131 Analytical Biochemistry 419 (1983), describe three (two fluorescent and one colored) derivatives of cadaverine which could serve as substrates for fibrinoligase (FXIIIa). Fluorescent and colorimetric techniques for measuring Factor XIII are described. Muszbek et al., 31 Clinical Chemistry 35-40 (1985), describe a kinetic assay for determination of Factor XIII in plasma. The release of ammonia in the FXIIIa reaction with a modified .beta.-casein is continuously monitored with an indicator reaction. U.S. Pat. No. 4,601,977 to Ogawa et al. "Method for Measuring the Activity of Plasma Factor XIII" (July 22, 1986) describes a method using incorporation of a fluorescent cadaverine derivative into casein, followed by use of a column gel for separating reacted cadaverine derivative from unreacted cadaverine derivative.
Despite the numerous methodologies described, there has been a continuing need for a simple assay procedure which is suitable for routine analysis for Factor XIII in the diagnostic laboratory.